Primer probe set, kit for simultaneously detecting multiple frog viruses and application thereof

Abstract

The application discloses a primer probe set for simultaneously detecting multiple frog viruses, a kit and application thereof, and belongs to the technical field of animal virus detection.The primer probe set for detecting frog viruses comprises a primer group with the sequence shown in SEQ ID NO.1-2 and a probe with the sequence shown in SEQ ID NO.3.According to the D5family NTPase gene sequence of the Ranavirus member, the primer and the probe are designed, and through optimization of reaction conditions, a method for detecting frog viruses based on qPCR is finally established.The method has the characteristics of high specificity, good repeatability, high sensitivity, simple operation and reliability, and has good market application prospect and popularization and application value.

Description

Technical Field

[0001] This invention relates to the field of animal virus detection technology, and in particular to a primer and probe set, a kit for the simultaneous detection of multiple frog viruses, and their applications. Background Technology

[0002] Frog viruses are novel pathogens that can infect reptiles, fish, and amphibians, with a host range encompassing 175 species of poikilothermic vertebrates from 52 families. They cause population declines in many fish and amphibian species, resulting in significant economic losses to the aquaculture industry. In China, annual losses from aquatic diseases caused by frog viruses reach billions of yuan.

[0003] Currently, seven species of the genus Ranavirus have been recognized by the International Committee on Taxonomy of Viruses (ICTV): Ranavirus ambystoma 1, Ranavirus alytes 1, Ranavirus perca 1, Ranavirus gadus 1, Ranavirus rana 1, Ranavirus micropterus 1, and Ranavirus epinephelus 1. Representative strains include Ambystoma tigrinum virus (ATV), common midwife toad virus (CMTV), epizootic haematopoietic necrosis virus (EHNV), cod iridovirus (CoIV), frog virus type 3 (FV3), largemouth bass virus (LMBV), and Singapore grouper iridovirus (SGIV). Current methods for identifying members of the Rhabdovirus genus include restriction endonuclease analysis (REA), viral protein mapping, and DNA sequence analysis. The Rhabdovirus genome contains 26 relatively conserved core genes, which are involved in viral transcription, replication, and structural function. Analysis of these 26 core genes revealed high similarity between the D5 family NTPase gene and the major capsid protein (MCP) gene. MCP is a structural protein, approximately 1300-1500 bp in length. The similarity of MCP among different frog virus genera is over 69%, and it is frequently used for frog virus diagnosis and identification. Meanwhile, the D5 family NTPase gene, approximately 2700-2900 bp in length, is involved in viral replication and repair, with a similarity of over 66%.

[0004] The D5 family NTPase gene is very similar to the MCP gene, but is much longer, making it a highly suitable gene for the diagnosis and identification of members of the frogvirus genus. Based on the sequence of the D5 family NTPase gene, members of the frog virus genus can be divided into three categories. The first category, represented by FV3, includes FV3-type frog viruses, such as epizootic haematopoietic necrosis virus (EHNV), frog virus type 3 (FV3), Andrias davidianus iridovirus disease (ADIV), soft-shelled turtleir idovirus (STIV), Bohle virus (BIV), Tiger frog virus (TFV), and Rana nigromaculata ranavirus (RNRV). The second category, represented by Santee-Cooperranavirus (SCRV), includes LMBV and Siniperca chuatsivirus. The third category is the SGIV class, represented by SGIV, which includes SGIV and GIV.

[0005] The World Organisation for Animal Health (WOAH) Manual of Diagnostic Methods for Aquatic Animal Diseases recommends a diagnostic method for frog viruses that involves PCR amplification of the MCP gene followed by restriction enzyme digestion analysis for FV3, BIV, European catfish virus (ECV), European sheatfish virus (ESV), and EHNV. However, this recommended method does not include analysis for SCRV virus. Li Huifang's quantitative real-time PCR method can detect members of the frog virus genus except for Singapore grouper iridovirus, but it does not analyze its ability to detect SCRV. Zhang Lifeng designed three primer pairs and three probes to identify members of the frog virus genus. Chinese patent document CN104099428A discloses a universal primer design for three types of frog viruses based on the MCP sequence, including three primer pairs and three probes, which can detect FV3, BIV, EHNV, ADIV, STIV, LMBV, and SGIV. Chinese patent documents CN 112301168A, CN118703703A, and CN 113293235A respectively disclose nucleic acid combinations, kits, and detection methods for detecting individual members of the frog virus genus. Specifically, CN 112301168A provides a quantitative real-time PCR detection method for LMBV, CN118703703A provides a quantitative real-time PCR detection method for SCRaV, and CN 113293235A provides a PCR detection method for FV3. Summary of the Invention

[0006] The purpose of this invention is to provide a primer-probe set, kit, and application for the simultaneous detection of multiple frog viruses, addressing the problems of complex identification methods, high workload, and limited methodologies in existing technologies. This invention designs a pair of primers and a probe targeting the D5 family NTPase gene for the detection and quantitative analysis of multiple frog viruses, specifically including FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV. It exhibits high sensitivity, with a detection limit of 10 copies / μL. This invention uses only a pair of specific primers and a probe to identify members of the frog virus genus, offering advantages such as high specificity, high sensitivity, simple operation, and high detection efficiency, demonstrating promising market application prospects and significant potential for widespread application.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] In one aspect, the present invention provides a primer-probe set for the simultaneous detection of multiple frog viruses, including an upstream primer, a downstream primer, and a probe;

[0009] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the probe is shown in SEQ ID NO.3.

[0012] Preferably, the 5' end of the probe sequence is modified with a fluorescent reporter group, and the 3' end of the probe sequence is modified with a fluorescent quencher group.

[0013] Preferably, the fluorescent reporter group includes FAM, and the fluorescent quencher group includes BHQ1.

[0014] Secondly, the present invention also provides a detection kit for simultaneously detecting multiple frog viruses, including the aforementioned primer and probe set.

[0015] Preferably, the test kit also includes PCR buffer, positive control and negative control.

[0016] Thirdly, the present invention also provides the application of the primer-probe set or the detection kit in the preparation of detection reagents for detecting frog viruses.

[0017] Fourthly, the present invention also provides a method for detecting various frog viruses for non-disease diagnostic purposes using the primer and probe set or the detection kit, characterized in that the method includes the step of using the DNA of the sample to be tested as a template and performing a qPCR reaction using the primer and probe set or the detection kit.

[0018] When the positive control shows an amplification curve and the negative control does not, if the test sample shows an amplification curve and the Ct value is ≤35, the frog virus nucleic acid in the test sample is determined to be positive; if the test sample does not show an amplification curve, the frog virus nucleic acid in the test sample is determined to be negative.

[0019] Preferably, the various frog viruses include FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV.

[0020] Preferably, the qPCR reaction system consists of: 10 μL PCR buffer, 0.4 μL each of forward and reverse primers, 0.2 μL probe, 2 μL DNA template, and ddH2O to a final volume of 20 μL.

[0021] Preferably, the qPCR reaction program is: 95℃, 15s, 62℃, 1min, 40 cycles.

[0022] The present invention discloses the following technical effects:

[0023] This invention selects conserved regions of the D5 family NTPase gene of frog viruses of the genera *Axolotl.*, *Midnight Toad.*, *Perch.*, *Cod.*, *Rana.*, *Black Bass.*, and *Grouper.* to design primers and probes. The purpose is to achieve accurate and rapid on-site detection of frog viruses, and it has the characteristics of simple, specific and efficient identification of multiple members of the frog virus genera.

[0024] The limit of detection of this invention can reach 10 copies / μL, effectively detecting FV3, SCRV, and SGIV frog viruses in samples, specifically including FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV. It also exhibits strong specificity for Koi herpesvirus (KHV), Tilapia Lake Virus (TiLV), viral nervous necrosis virus (VNNV), channel catfish virus (CCV), carp edemavirus disease (CEV), infectious hematopoietic necrosis virus (IHNV), infectious spleen and kidney necrosis virus (ISKNV), and Grass carp virus type II. There was no cross-reactivity between reovirus (GcRV-2) and herpesvirus type II (Cyprinidherpesvirus II, CyHV-2). Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The results are for detection at different reaction temperatures according to the present invention;

[0027] Figure 2 The results show the detection results of different primer concentrations in this invention;

[0028] Figure 3 The results are for different probe concentrations according to the present invention.

[0029] Figure 4 This invention is specific to the sample detection results;

[0030] Figure 5 This is the result of the SGIV template concentration sensitivity detection in this invention;

[0031] Figure 6 This is the result of the LMBV template concentration sensitivity detection in this invention;

[0032] Figure 7 This is the result of the FV3 template concentration sensitivity detection in this invention. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] SEQ ID NO.1: 5'-CTGTTTGAGAARATGCTGGG-3';

[0039] SEQ ID NO.2: 5'-CCGCGTTTATGGTYTCGTC-3';

[0040] SEQ ID NO.3: 5'-TCCATCACKGCCCACCTCACTCC-3';

[0041] R, Y, and K are all degenerate bases, where R refers to G or A, Y refers to C or T, and K refers to G or T.

[0042] Example 1: Design and Synthesis of qPCR Primers and Probes

[0043] Primers and probes were designed based on the NTPase sequence characteristics of the D5 family of frogvirus members in GeneBank.

[0044] Using the D5 family NTPase genes of FV3 (MH351268), BIV (NC_038507.1), EHNV (MT510734), TFV (MT512504.1), LMBV (FR682503.1), SCRaV (OQ267588), SGIV (AY521625), ADIV (KC865735.1), STIV (EU627010.1), and RNRV (MG791866.1) from GeneBank as detection targets, qPCR primers and probes for the genus *Ranavirus* were designed using Oligo 7 software. The primers include a forward primer with the sequence shown in SEQ ID NO.1 and a reverse primer with the sequence shown in SEQ ID NO.2. The sequence of the specific probe is shown in SEQ ID NO.3, where R, Y, and K are degenerate bases, R represents G or A, Y represents C or T, and K represents G or T.

[0045] The probe SEQ ID NO.3 is modified with a FAM fluorescent reporter group at 5' and a BHQ1 fluorescent quencher group at 3'.

[0046] The primers and probes mentioned above were all synthesized by BGI Genomics Co., Ltd.

[0047] Example 2: qPCR detection method for members of the Frogvirus genus

[0048] Using the genomic DNA of the sample as a template, qPCR was performed using the primers and probes designed in Example 1, and the fluorescence signal was detected. The specific steps are as follows:

[0049] The qPCR reaction system consisted of: 10 μL of real-time PCR buffer, 0.4 μL each of forward and reverse primers, 0.2 μL of probe, and 2 μL of DNA template, with ddH2O added to a final volume of 20 μL. After mixing and centrifugation, the reaction tube was immediately placed in a real-time PCR instrument to begin the qPCR reaction. The reaction conditions were: 95℃ for 15 s, 62℃ for 1 min, for 40 cycles; the corresponding fluorescence signal was acquired during the annealing and extension phase at 62℃.

[0050] When the positive control shows an amplification curve and the negative control does not, if the test sample shows an amplification curve and the Ct value is ≤35, the frog virus nucleic acid in the test sample is determined to be positive; if the test sample does not show an amplification curve, the frog virus nucleic acid in the test sample is determined to be negative.

[0051] Example 3: Optimization of qPCR reaction conditions for members of the Frogvirus genus

[0052] Genomic DNA was extracted from SGIV and amplified using primers SEQ ID NO.1 and SEQ ID NO.2. The PCR product was purified and cloned into the pM19-T vector to construct the recombinant plasmid, named pMD19-SGIV. After PCR verification and sequencing confirmation, the concentration of the recombinant plasmid was determined using a full-wavelength reader. The formula was: Plasmid copy number (copies / μL) = 6.02 × 10⁻⁶. 23 (Copies / mol) × plasmid mass concentration (g / μL) / plasmid relative molecular mass (g / mol) is used to convert the mass concentration of the recombinant plasmid standard to the copy number concentration.

[0053] Recombinant plasmid pMD19-SGIV diluted to 10 6 copy / μL-10 3 Using copies / μL as a template, the optimal reaction conditions for the qPCR detection method were established by optimizing primer concentration, probe concentration, and reaction temperature.

[0054] The qPCR reaction system consisted of: 10 μL of real-time PCR buffer, 0.4 μL each of forward and reverse primers, 0.2 μL of probe, and 2 μL of DNA template, with ddH2O added to a final volume of 20 μL. After mixing and centrifugation, the reaction tube was immediately placed in a real-time PCR instrument to begin the qPCR reaction. The reaction conditions were: 95℃ for 15 s, 62℃ for 1 min, for 40 cycles; the corresponding fluorescence signal was acquired during the annealing and extension phase at 62℃.

[0055] qPCR reactions were performed at fixed probe concentrations of 0.2 μmol / μL and primer concentrations of 0.4 μmol / μL, at reaction temperatures of 57℃, 58℃, 59℃, 60℃, 61℃, and 62℃ to screen for the optimal reaction temperature. Results showed that qPCR reactions could be performed at all temperatures: 57℃, 58℃, 59℃, 60℃, 61℃, and 62℃. (See also...) Figure 1 .

[0056] qPCR reactions were performed at annealing temperature of 62℃ with a fixed probe concentration of 0.2 μmol / μL and primer concentrations of 0.1 μmol / μL, 0.2 μmol / μL, 0.3 μmol / μL, 0.4 μmol / μL, 0.5 μmol / μL, and 0.6 μmol / μL to screen for the optimal primer concentration. The results showed that qPCR reactions could be performed at primer concentrations ranging from 0.1 μmol / μL to 0.6 μmol / μL. (See [link to relevant documentation]). Figure 2 .

[0057] qPCR reactions were performed at annealing temperature of 62℃ with a fixed primer concentration of 0.4 μmol / μL and probe concentrations of 0.05 μmol / μL, 0.1 μmol / μL, 0.15 μmol / μL, 0.2 μmol / μL, 0.25 μmol / μL, and 0.3 μmol / μL to screen for the optimal probe concentration. The results showed that qPCR reactions could be performed at probe concentrations ranging from 0.05 μmol / μL to 0.3 μmol / μL. (See [link to relevant documentation]). Figure 3 .

[0058] Example 4: Specificity analysis of qPCR detection method for members of the frog virus genus

[0059] Based on the above experimental optimization results, primers and probes were used to perform qPCR reactions on nine common fish viruses (KHV, TiLV, VNNV, CCV, CEV, IHNV, ISKNV, GcRV-2, and CyHV-2) and ten frog virus genera (FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV). The results showed that only frog virus genera members exhibited amplification curves. (See [link to relevant documentation]). Figure 4 .

[0060] Example 5: Sensitivity Analysis of qPCR Detection Method for Frog Virus Members

[0061] Genomic DNA of LMBV and FV3 viruses was extracted, and standard plasmids were constructed according to the method in Example 3, named pMD19-LMBV and pMD19-FV3, respectively. Plasmids pMD19-SGIV, pMD19-LMBV, and pMD19-FV3 were serially diluted 10-fold to adjust the concentration to 10⁻⁶. 6 The concentration of primers was 0.4 μmol / μL, the probe concentration was 0.2 μmol / μL, and the reaction conditions were 95℃ for 15 s, 62℃ for 1 min (40 cycles). The results showed that the limit of detection for this method was 10 copies / μL. (See [link to relevant documentation]). Figures 5-7 .

[0062] Example 6: Repeatability Analysis of qPCR Detection Method for Frog Virus Members

[0063] Plasmids pMD19-SGIV, pMD19-LMBV, and pMD19-FV3 were serially diluted 10-fold to adjust the concentration to 10. 6 copy / μL-10 4 The sample was tested three times by three different researchers using the method described in Example 2. The standard deviation (SD) and coefficient of variation (CV) of the Ct values ​​between and within groups were used as the judgment criteria. The results are shown in Table 1.

[0064] Table 1. Repeatability analysis of qPCR detection methods

[0065]

[0066] As shown in the table above, the qPCR detection method established in this invention has an intra-group coefficient of variation of less than 2.76% and an inter-group coefficient of variation of less than 3.83%, indicating that the real-time fluorescence quantitative PCR detection method established in this application has good repeatability.

[0067] Example 7 Verification Experiment

[0068] From 2024 to 2025, 42 ornamental fish samples and 6 frog samples with suspected positive results were collected. The samples were tested simultaneously using the conventional PCR method recommended by WOAH and the qPCR method of Example 2. The results showed that the conventional PCR method detected 19 positive samples and the qPCR method detected 42 positive samples. The positive concordance rate of the two detection methods was 100%, and the detection rate of the qPCR method was higher than that of the conventional PCR detection method.

[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a primer-probe set or a detection kit containing said primer-probe set in the preparation of a detection reagent for detecting frog viruses, characterized in that, The primer-probe set includes an upstream primer, a downstream primer, and a probe; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; The nucleotide sequence of the probe is shown in SEQ ID NO.3; The frog viruses include FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV.

2. A method for detecting various frog viruses for non-disease diagnostic purposes using the primer and probe set or the detection kit described in claim 1, characterized in that, The method includes the steps of using the DNA of the sample to be tested as a template and performing a qPCR reaction using the primer and probe set or the detection kit. When the positive control shows an amplification curve and the negative control does not, if the test sample shows an amplification curve and the Ct value is ≤35, the frog virus nucleic acid in the test sample is determined to be positive; if the test sample does not show an amplification curve, the frog virus nucleic acid in the test sample is determined to be negative. The various frog viruses mentioned include FV3, BIV, EHNV, ADIV, STIV, LMBV, SGIV, SCRaV, TFV, and RNRV.

3. The method according to claim 2, characterized in that, The qPCR reaction system consisted of: 10 μL PCR buffer, 0.4 μL each of forward and reverse primers, 0.2 μL probe, 2 μL DNA template, and ddH2O to a final volume of 20 μL.

4. The method according to claim 2, characterized in that, The qPCR reaction program was: 95℃, 15s, 62℃, 1min, 40 cycles.

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